Folding and Duplex Formation in Sequence-Defined Aniline Benzaldehyde Oligoarylacetylenes

In all known genetic polymers, molecular recognition via hydrogen bonding between complementary subunits underpins their ability to encode and transmit information, to form sequence-defined duplexes, and to fold into catalytically active forms. Reversible covalent interactions between complementary subunits provide a different way to encode information, and potentially function, in sequence-defined oligomers. Here, we examine six oligoarylacetylene trimers composed of aniline and benzaldehyde subunits. Four of these trimers self-pair to form two-rung duplex structures, and two form macrocyclic 1,3-folded structures. The equilibrium proportions of these structures can be driven to favor each of the observed structures almost entirely depending upon the concentration of trimers and an acid catalyst. Quenching the acidic trimer solutions with an organic base kinetically traps all species such that they can be isolated and characterized. Mixtures of complementary trimers form exclusively sequence-specific 3-rung duplexes. Our results suggest that reversible covalent bonds could in principle guide the formation of more complex folded conformations of longer oligomers.

. Relevant section of the 1 H NMR spectra showing the conversion of ss-BAB to ds-BAB at different [BAB] after treatment with TFA (2 mM) and quenching with TEA (6 mM). The CHN imine peaks for ds-BAB, the CHO aldehyde peaks for ds-BAB and ss-BAB, and the aromatic peaks from the A subunit of ss-BAB are labeled as such.  Figure S2. Relevant section of the 1 H NMR spectra showing the conversion of ss-BAB to ds-BAB after treatment with different amounts of TFA. The CHN imine peaks for ds-BAB, the CHO aldehyde peaks for ds-BAB and ss-BAB, and the aromatic peaks from the A subunit of ss-BAB are labeled as such.  Figure S4. Relevant section of the 1 H NMR spectra showing the conversion of ss-ABA to ds-ABA after treatment with different amounts of TFA and after quenching the reaction with a 3-fold excess of TEA relative to the [TFA]. The CHN imine peaks for ds-ABA and the CHO aldehyde peaks for ss-ABA are labeled as such.  2M+Na/+1 S19 Figure S18. ESI-TOF Mass spectra for ds-, and fold-ABOs. a These peaks are distinguishable from the corresponding fold-ABOs by their isotopic ratios indicating a +2 charge state. b These peaks are not distinguishable from the M+Na/+1 ions of the corresponding ds-ABOs, however NMR data confirms these solutions do not contain significant amounts of ds-ABO and so it can be concluded that these peaks arise from 2M+Na/+1 ions of the fold-ABO.  . Concentration dependence of the 1 H NMR chemical of shift of 3-rung duplexes in CDCl3 at rt and 50 °C for three resonances: blue, the internal imine (NHC), red the terminal imines (NHC), and yellow the aromatic protons meta to the ester functional groups. Curves represent the line of best fit to the equal K model of indefinite association. The error is the standard error estimated from the variance-covariance matrix of the least-squares fitted parameters.

EXPERIMENTAL SECTION
General. All solvents and catalysts, as well as starting materials 3-ethnylbenzlaldhye 1 and 3-ethnylaniline 4 were purchased from commercial sources and used without further treatment. Diiodide 2, [1] 3,5-diethnylbenzalehyde, [2] and 3,5-diethnylaniline [3] were prepared via previously reported methods. "Dry" CDCl3 was prepared by treating and storing commercially available CDCl3 over 4 Å molecular sieves. THF was purchased as a solution stabilized with 250 ppm BHT and fractionally distilled before use. Removal of peroxides from the THF was crucial to the solution stability of the ABOs. MM2 energy minimum calculations for 1,3-folded AAB and 1,2-folded AAB were accomplished using the Perkin Elmer Chem3D software package. 1 H NMR and 13 C NMR spectra were recorded at 93.94 kG ( 1 H 400 MHz, 13 C 100 MHz) at 25 °C. Hydrogen chemical shifts are expressed in parts per million (ppm) relative to the residual protio-solvent resonance: CDCl3 δ 7.26. For 13 C spectra, the centerline of the solvent signal was used as internal reference: CDCl3 δ 77.16. Unless otherwise noted, each carbon resonance represents a single carbon (relative intensity).
ESI-TOF high resolution mass spectrometric data were obtained on a ToF (time-of-flight) Agilent Technologies system. Samples were injected as 10 uM solutions in 8:2 THF:H2O with 20 uM sodium acetate. The MS settings were: capillary voltage 4500 V, desolvation temperature 300 °C, fragmentor voltage 450 V. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry experiments were carried out as follows: 0.5 uL of a 100 uM solution of 2,5-dihydroxybenzoic acid matrix (Sigma) in methanol was deposited on a MTP 384 polished steel BC target plate (Bruker), and the solvent was allowed to evaporate. A single 0.5 μL aliquot of analyte in CDCl3 was then added on top of the 2,5-dihydroxybenzoic acid film and the sample analyzed with an autoflextreme MALDI-TOF mass spectrometer (Bruker). For Ke determination, a global fit of three protons to the equal K model of indefinite association was preformed using the MATLAB software package the multiple curve fitting with common parameters tool. [4]

ss-AAA.
To a 20 mL scintillation vial AsPh3 (0.026 mmol, 7.9 mg) and Pd2(dba)3 (0.0033 mmol, 3.0 mg, 10 mol%) were added and dissolved in anhydrous toluene (0.5 mL). The head space of the vial was purged with argon then the vial sealed with a screw on cap. The purple solution was gently swirled until the Pd2(dba)3 dissolved and the color changed to bright yellow (approx. 10 min). In a separate vial equipped with a stir bar, 2,6-diethynylaniline (9.2 mg, 0.065 mmol, 1 equiv.) and aryl-iodide 5 (2 equiv., 60 mg, 0.13 mmol) were dissolved in anhydrous toluene (1.3 mL) and DIPEA (12 equiv., 1.6 mmol, 271 uL). While stirring under a stream of argon the catalyst solution was added dropwise to the solution containing 2,6-diethynylaniline and 5. The vessel was sealed with a screw on cap and heated to 40 °C. After stirring overnight, the reaction was cooled to rt, transferred to a round bottom flask, and diluted with DCM (approx. 50 mL) and TEA (approx. 1 mL). Silica gel (approx. 2 g) was added to the vessel and the crude reaction mixture was concentrated onto the silica gel. Silica gel flash chromatography (hexane with a 70-100% gradient of EtOAC over 5 minutes, then held at 100% EtOAc for 25 min, both organic solvents were basified with 0.1 % TEA) gave ss-AAA (34 mg, 0.042 mmol, 65%) as a white solid after concentration in vacuo. 1

ss-BBB.
To a 20 mL scintillation vial AsPh3 (0.19 mmol, 19 mg) and Pd2(dba)3 (0.0075 mmol, 6.8 mg, 5 mol%) were added and dissolved in anhydrous toluene (1.0 mL). The head space of the vial was purged with argon then the vial sealed with a screw on cap. The purple solution was gently swirled until the Pd2(dba)3 dissolved and the color changed to bright yellow (approx. 10 min). In a separate vial equipped with a stir bar, 2,6-diethynylbenzaldehyde (19 mg, 0.12 mmol, 1.0 equiv.) and aryl-iodide 3 (2.5 equiv., 143 mg, 0.3 mmol) were dissolved in anhydrous toluene (3.0 mL) and DIPEA (12 equiv., 1.5 mmol, 261 uL). While stirring under a stream of argon the catalyst solution was added dropwise to the solution containing 2,6-diethynylbenzaldehyde and 3. The vessel was sealed with a screw on cap and stirred at rt. After stirring overnight, the crude reaction mixture was transferred to a round bottom flask and diluted with DCM (approx. 50 mL) and TEA (approx. 1 mL). Silica gel (approx. 5 g) was added to the vessel and the crude reaction mixture was concentrated onto the silica gel. Silica gel flash chromatography (hexane with a 20-90% gradient of EtOAC) gave pure ss-BBB (98 mg, 0.11 mmol, 94%) as a white solid after concentration in vacuo. 1

ss-ABA.
To a 20 mL scintillation vial AsPh3 (0.02 mmol, 6.2 mg) and Pd2(dba)3 (0.0025 mmol, 2.2 mg, 10 mol%) were added and dissolved in anhydrous toluene (0.5 mL). The head space of the vial was purged with argon then the vial sealed with a screw on cap. The purple solution was gently swirled until the Pd2(dba)3 dissolved and the color changed to bright yellow (approx. 10 min). In a separate vial equipped with a stir bar, 2,6-diethynylbenzaldehyde (7.7 mg, 0.05 mmol, 1 equiv.) and aryl-iodide 5 (2 equiv., 47 mg, 0.1 mmol) were dissolved in anhydrous toluene (1 mL) and DIPEA (12 equiv., 0.6 mmol, 104 uL). While stirring under a stream of argon the catalyst solution was added dropwise to the solution containing 2,6-diethynylbenzaldehyde and 5. The vessel was S32 sealed with a screw on cap and heated to 40 °C. After stirring at overnight, the reaction was cooled to rt, transferred to a round bottom flask, and diluted with DCM (approx. 50 mL) and TEA (approx. 1 mL). Silica gel (approx. 2 g) was added to the vessel and the crude reaction mixture was concentrated onto the silica gel. Silica gel flash chromatography (hexane with a 70-100% gradient of EtOAC over 5 minutes, then held at 100% EtOAc for 10 min, both organic solvents were basified with 0.1 % TEA) gave ss-ABA (31 mg, 0.037 mmol, 74%) as a white solid after concentration in vacuo. 1

ds-ABA.
A 7.5 mM solution of ss-ABA (3.7 mg, 4.5 µmol) in dry CDCl3 (600 uL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-ABA was acquired and then 10 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The capped NMR tube was immediately inverted several times to mix, and then the solvent evaporated under a stream of Ar. Dry CDCl3 (600 uL) was added to the NMR tube and the yellow residue allowed to dissolve (approx. 5 min). Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (0.5 uL) directly to the NMR tube. Another 1 H NMR spectrum was acquired showing complete conversion to ds-ABA. Using DCM (~4 mL) the solution was transferred to a test tube and the organic layer washed with water (4 mL) 3 times. The cloudy organic layer was then concentrated in vacuo to give ds-ABA (3.7  A 7.5 mM solution of ss-BAB (7.6 mg, 9 µmol) in dry CDCl3 (1.2 mL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-BAB was acquired and then 10 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The capped NMR tube was immediately inverted several times to mix, and then the solvent evaporated under a stream of Ar. Dry CDCl3 (1.2mL) was added to the NMR tube and the yellow residue allowed to dissolve (approx. 5 min). Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (1 uL) directly to the NMR tube. Another 1 H NMR spectrum was acquired showing nearly complete conversion to ds-BAB. Using DCM (~4 mL) the solution was transferred to a test tube and the organic layer washed with water (4 mL) 3 times. The cloudy organic layer was then concentrated in vacuo to give ds-BAB (7.

ds-AAB.
A 7.5 mM solution of ss-AAB (3.7 mg, 4.5 umol) in dry CDCl3 (600 uL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-AAB was acquired and then TFA was added (2 uL). The capped NMR tube was immediately inverted several times to mix.
Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (6 uL) directly to the NMR tube. Another 1 H NMR spectrum was acquired showing nearly complete conversion to ds-AAB. Using DCM (~4 mL) the solution was transferred to a test tube and the organic layer washed with water (4 mL) 3 times. The cloudy organic layer was then concentrated in vacuo to give ds-AAB (3.7 mg, 2.3 µmol, 100%) as a white solid. 1

ds-ABB.
A 7.5 mM solution of ss-ABB (7.6 mg, 9 umol) in dry CDCl3 (1.2 mL) was prepared in an NMR tube. An initial 1 H NMR spectrum of ss-ABA was acquired and then 10 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The capped NMR tube was immediately inverted several times to mix, and then the solvent evaporated under a stream of Ar. Dry CDCl3 (600 uL) was added to the NMR tube, 5 uL of a solution of 1% TFA in CDCl3 (v/v) was added, and the yellow residue allowed to dissolve (approx. 5 min). Another 1 H NMR spectrum was acquired, and then the acidic solution was quenched by adding TEA (1 uL) directly to the NMR tube.

fold-AAB.
A 3.9 mM solution of ss-AAB (6.4 mg, 7.7 umol) in dry CDCl3 (2 mL) was prepared in a 100 mL flask, and then a 5 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The solvent was evaporated under a stream of Ar, then the residue was dissolved in 80 mL dry CDCl3 (100 uM AAB), and then 5 uL of a solution of 1% TFA in CDCl3 (v/v) was added. The solution was allowed to stand for 5 min and then the acid was quenched with 6 uL TEA. An 1 H NMR spectrum of an aliquot of the solution showed complete